Polysaccharide degrading enzymes are useful in a variety of applications, such as in animal feed, industrial applications, and, in particular, in ethanol production.
Fossilized hydrocarbon-based energy sources, such as coal, petroleum and natural gas, provide a limited, non-renewable resource pool. Because of the world's increasing population and increasing dependence on energy sources for electricity and heating, transportation fuels, and manufacturing processes, energy consumption is rising at an accelerating rate. The US transportation sector alone consumes over 100 billion gallons of gasoline per year. Most (˜60%) of the oil used in the US today is imported, creating a somewhat precarious situation in today's political climate because supply disruptions are highly likely and would cripple the ability of the economy to function. Fossil petroleum resources, on which our standard of living currently depends, will likely be severely limited within the next 50-100 years.
The production of ethanol from lignocellulosic biomass can utilize large volumes of agricultural resources that are untapped today. Ethanol is key to partially replacing petroleum resources, which are limited. Ethanol fuels burn cleanly and because of this, ethanol replacement of petroleum fuels at any ratio will have a positive impact on the environment. Production of ethanol from domestic, renewable sources also ensures a continuing supply. For these reasons, the production of ethanol fuels from lignocellulosic biomass are being developed into a viable industry. High yields of glucose from cellulose (using cellulase enzymes) are required for any economically viable biomass utilization strategy to be realized. The US is one country involved in ethanol production and currently manufactures approximately over three billion gallons of ethanol from corn grain-derived starch. (American Coalition of Ethanol Production, www.ethanol.org; also, Sheehan, J. “The road to bioethanol: A strategic perspective of the US Department of Energy's National Ethanol Program” Himmel M E, Baker J O, Saddler J N eds., Glycosyl Hydrolases for Biomass Conversion, 2-25). Ethanol that is produced from corn starch, however, has not been cost-effective alternative to fossil fuels.
Unharvested residues from agricultural crops are estimated at a mass approximately equal to the harvested portion of the crops. Specifically for the corn crop, if half of the residue could be used as a feedstock for the manufacture of ethanol, then about 120 million tons of corn stover would be available annually for biomass conversion processes (Walsh, Marie E. Biomass Feedstock Availability in the United States. State Level Analysis. 1999). Assuming that mature, dry corn stover is approximately 40% cellulose on a dry weight basis then 48 million tons of cellulose/year would be available for hydrolysis to glucose. Using today's technology, a ton of cellulose will yield approximately 100 gallons of ethanol.
Because known technologies for ethanol production from plant biomass have been more costly than the market price for ethanol, ethanol will not become an important alternative to fossil fuels, unless the price of fossil fuels rises substantially. If, however, the cost of the production of ethanol from plant biomass could be reduced, then ethanol might become a cost-effective alternative to fossil fuels even at today's prices for fossil fuels.
Plant biomass is a complex matrix of polymers comprising the polysaccharides cellulose and hemicellulose, and a polyphenolic complex, lignin, as the major structural components. Any strategy designed to substitute lignocellulosic feedstocks for petroleum in the manufacture of fuels and chemicals must include the ability to efficiently convert the polysaccharide components of plant cell walls to soluble, monomeric sugar streams. Cellulose, the most abundant biopolymer on earth, is a simple, linear polymer of glucose. However, its semi-crystalline structure is notoriously resistant to hydrolysis by both enzymatic and chemical means. Yet, high yields of glucose from cellulose are critical to any economically viable biomass utilization strategy.
Nature has developed effective cellulose hydrolytic machinery, mostly microbial in origin, for recycling carbon from plant biomass in the environment. Without it, the global carbon cycle would not function. To date, many cellulase genes have been cloned and sequenced from a wide variety of bacteria, fungi and plants, and many more certainly await discovery and characterization (Schulein, M, 2000. Protein engineering of cellulases. Biochim. Biophys. Acta 1543:239-252); Tomme P, et al. 1995. Cellulose Hydrolysis by Bacteria and Fungi. Advances in Microbial Physiology 37:1-81). Cellulases are a subset of the glycosyl hydrolase superfamily of enzymes that have been grouped into at least 13 families based on protein sequence similarity, enzyme reaction mechanism, and protein fold motif.
The economics of using corn stover or any other source of lignocellulosic biomass to produce ethanol is ominous at best and is the limiting step behind the attainment of such a goal. The current cost of making ethanol from any source of lignoceliulosic biomass with the current enzyme production systems and the biomass collection and pretreatment technology is in the order of about $1.50 per gallon. This is due to the high operation costs of collecting and transporting the lignocellulosic raw material to destination plants, producing the polysaccharide-degrading enzymes and the high cost of pretreating the lignocellulosic raw material to facilitate its enzymatic degradation. To become economical, the processes for ethanol production have to be integrated into the cultivation of agricultural crops. In particular, the process of producing the enzymes required for ethanol production as well as the collection of lignocellulosic raw material have to be integrated into the normal operations of crop cultivation. The crop market will generate the revenues necessary to economically justify its cultivation and the production of ethanol will be a by-product of this operation.
At present enzyme production is primarily by submerged culture fermentation. The scale-up of fermentation systems for the large volumes of enzyme required for biomass conversion would be difficult and extremely capital intensive. For purposes of comparison, a single very large (1 million liter), aerobic fermentation tank could produce 3,091 tons of cellulase protein/yr in continuous culture. Currently, however, fermentation technology is practiced commercially on a significantly smaller scale and in batch mode, so production capacities are closer to 10% of the theoretical 3,091 tons calculated above. Thus, using these assumptions, current practices would yield 3000 times less than the 1.2 MM tons of enzyme needed to convert the cellulose content from 120 MM tons per year of corn stover. Capital and operating costs of such a fermentative approach to producing cellulases are likely to be impractical due to the huge scale and capital investment that will be required.
Several recombinant systems are available for protein production. Foreign proteins have been produced in animal cell cultures and transgenic animals. However, these methods are very expensive and time intensive, particularly in the scale-up of cultures or herds large enough for industrial enzyme production, making them highly impractical. Bacteria and fungi are relatively simple systems but require a large initial investment for capital equipment. On the other hand, crop-based production systems may offer an attractive and cost-effective alternative for industrial enzyme production at the scale required for biomass conversion. Transgenic plants require the lowest capital investment (mainly for dedicated harvesting equipment and storage) of all production systems. The cost of producing crude recombinant protein in plants could be three orders of magnitude lower than that of the mammalian cell system, and 10 fold less than microbial fermentation (Elizabeth E. Hood and Susan L. Woodard. Industrial Proteins Produced from Plants. Molecular Farming. 2002. In: Plants as Factories for Protein Production. E E. Hood and J A Howard, Eds., Kluwer Academic Publishers, Dordrecht, The Netherlands pp. 119-135). Advantages of plant systems include the low cost of growing a large biomass, easy scale-up (increase of planted acreage), natural storage organs (tubers, seeds), and established practices for efficient harvesting, transporting, storing and processing of the plant.
Plant systems have been used to express polysaccharide degrading cellulases specifically with varying amounts of success (Table 1). Ziegler et al. (Ziegler, M T, et al. 2000, Accumulation of a thermostable endo-1,4-β-D-glucanase in the apoplast of Arabidopsis thaliana leaves. Molecular Breeding 6:37-46) have expressed an endoglucanase in Arabidopsis leaves and in tobacco tissue culture cells at high levels, but both systems are impractical for commercialization. In addition, some preliminary work has been done with potato (Dai Z, et al. 2000. Improved plant-based production of E1 endoglucanase using potato: expression optimization and tissue targeting. Molecular Breeding 6:277-285) but expression levels were relatively low. Studies with tobacco, alfalfa and potato leaves have shown that individual cellulase enzymes can be expressed in these plants (Ziegelhoffer T, et al. 1999. Expression of bacterial cellulase genes in transgenic alfalfa (Medicago sativa L.), potato (Solanum tuberosum L.) and tobacco (Nicotiana tabacum L.). Molecular Breeding 5:309-318; and U.S. Pat. No. 5,981,835) although not at levels that would allow economic production of the enzymes.
TABLE 1Examples of heterologous cellulase expression in plants and production considerations.TransgenicExpressionStableEnzymeGene sourceplant systemlevelstorageScalability4Endo-1,4-β-D-BacterialArabidopsis 26% TSPNo−glucanase(Acidothermus)(cell wallin leaves1targeted)Endo-1,4-β-D-BacterialPotato2.6% TSP2No+glucanase(Acidothermus)(cell wall orin leaveschloroplasttarget)Endo-1,4-β-D-BacterialAlfalfa~0.01% TSP3 No++glucanase(Thermonospora)(cytosolicin leavescytosoliclocalization)localizationTobacco0.1% TSP3No+(cytosolicin leaveslocalization)CellobiohydrolaseBacterialAlfalfa0.02% TSP3 No++(T. fusca)(cytosolicin leaveslocalization)Tobacco0.002% TSP3 No+(cytosolicin leaveslocalization)1Zeigler et al., 2000;2Dai et al., 2000;3Ziegelhoffer et al., 1999 and ~% TSP assumes 10% of leaf weight is soluble protein;4Scalability defined by 2002 US crop acreage, scale-up potential: −, unscalable; +, fair; ++, moderate; +++, significant.TSP = Total soluble protein.
None of the expression systems to date have shown a practical application of producing cellulases. In some of the examples the expression level is much too low to be of any commercial use. The highest level of expression achieved was in Arabidopsis. However, the use of this plant is impractical for commercial production of enzymes. It is a model organism, used because of its ease in transformation, but grows to a height of only three inches and could not possibly produce adequate amounts of enzyme for commercial purposes. The volume of material needed and the expression levels need to be such that commercial production is practicable. In general, expression levels should be at least about 0.1% of total soluble protein of the plant tissue used. None of the work to date has involved expression of cellulases in corn (Zea mays, L.). While the possiblity of expressing an enzyme to a particular organelle has been presented, and in one instance targeted to the chloroplast (See U.S. Pat. No. 6,429,359) success in increasing expression by targeting specific organelles in plants cells or secreting from cell wall has not been shown. Further, for plant production of the enzymes to be commercially viable, expression at commercial levels in a plant that can be grown, harvested and scaled to commercial quantities must be achieved on a reliable, consistent basis.
Combining these improvements with harvest methods that allow the simultaneous recovery of corn stover and corn grain by a single pass through the field reduces the cost of collecting the lignocellulosic raw material. Such single pass (also referred to as one-pass) harvesting cuts down on the number of times that farm machinery are driven through the fields. This approach minimizes soil compaction, reduces the amount of time invested in material collection and curtails the cost of fossil fuel and labor needed for operating the farm machinery. One-pass harvest is being developed by several groups, for example at Iowa State University by Dr. Graeme Quick. See records and minutes of the “Corn Stover Harvesting Field Demonstration and Biomass Harvesting Colloquium”, Harlan, Iowa. Oct. 29, 2001.
Provided by the invention are cost-effective methods for the saccharification of polysaccharides in crop residues. The methods of the invention find particular use in the integration of current practices for the cultivation of crop plants for the purpose of obtaining a commercially desired plant material with the production of commercial levels of polysaccharide degrading enzymes in the tissues of the crop plants and the use of the crop plant residues as a source of lignocellulosic biomass for the production of fermentable sugars.
The methods of the invention find use in transforming crop plants with a nucleotide sequence encoding at least one polysaccharide degrading enzyme, such as those degrading cellulose, hemicellulose or pectin. Any plant tissue expressing the enzyme can be the source of the enzyme. In one embodiment of the invention the same plant used to make the enzyme can be the source of the lignocellulose. The enzymes can be produced in any part of the plant (leaves, seed, roots, etc.) and used for subsequent treatment in degrading polysaccharides of the plant. In an embodiment the crop plant is a plant that produces seeds. The source of the enzyme preferably can be seed tissue, such as one or more of whole seed, hulls, seed coat, endosperm, or embryo (germ). More preferrably the seeds have a germ that is capable of being fractioned from the rest of the seed (the term degerminated is sometimes used when referring to separation of the germ) in a commercial milling process. In a preferred embodiment of the invention the enzyme(s) are expressed in the germ portion of the seed. In another preferred embodiment the level of enzymes that are produced in the germ portion of the the seed are at least about 0.1% of the dry weight of the seed.
In particular, the methods of the invention further provide a cost-effective integrated approach to producing fermentable sugars from corn stover that encompasses the production of polysaccharide degrading enzymes in the seeds of genetically engineered corn plants. A portion of or all of the seed can be the source of the degrading enzyme with other plant parts used for other purposes. The option is available to use a select tissue of the seed for commercial purpose, and other tissue used as the source of enzyme for the saccarification process. For example, the corn endosperm can be used as a source of starch, corn stover from the engineered plants as lignocellulosic biomass and embryo as the enzyme source. Further economic advantages are obtained in harvesting the seeds in a first operation and the stover in a second operation such that both operations are carried out concurrently by employing single-pass harvest operations.
The methods of the invention involve producing one or more cell wall polysaccharide-degrading enzymes in a crop plant by transforming the plant with at least one nucleotide construct comprising a nucleotide sequence encoding a cell wall polysaccharide-degrading enzyme operably linked to a promoter that drives expression in the crop plant, more preferably in the crop plant seed or a portion thereof, such that the production of the commercially desired plant material is not forfeited by the production of the enzymes.
The methods further involve obtaining from the transformed plant, tissue that expresses the cell wall polysaccharide-degrading enzyme or enzymes, contacting lignocellulosic biomass with this plant tissue, and exposing the combination to conditions that are favorable for the degradation of cell wall polysaccharides into fermentable sugars. The fermentable sugars can then be utilized for the production of ethanol or other desired molecules using fermentation procedures that are known in the art.
The inventors have devised an integrated method for the economic saccharification of lignocellulosic biomass and its conversion into ethanol. It is, therefore, an object of the present invention to provide cost-effective methods for converting polysaccharides in lignocellulosic biomass into fermentable sugars. It is also an object of the present invention to genetically engineer plants to produce cell wall degrading enzymes at commercially high levels and use such enzymes in saccharification of polysaccharides. A still further object is to obtain both the source of polysaccharides and source of enzymes from one crop. Another object of the invention is to integrate efficient harvest methods such as single pass harvest with the genetic engineering of corn plants to cost effectively produce ethanol from corn stover. A further object of the invention is to produce commercially acceptable levels of polysaccharide-degrading enzymes in corn plants. Yet another object of the invention is to target the expression of polysaccharide-degrading enzymes to corn seeds, preferably to the germ portion of the seed.
In one embodiment of the invention, production of recombinant cellulases in plants is provided that improves over prior attempts to express cellulases in plants in reliability of enzyme production and at commercial levels.
In an embodiment of the invention cellulases are produced in corn plants.
Another object of the invention is the application of large-scale production of cellulases to industrial markets for which it had previously been economically unfeasible to enter.
In yet another embodiment of the invention the cellulases are preferentially expressed to the seed of the plant.
In an embodiment of the invention expression of cellulases is targeted to specific locations within the plant cell in order to increase expression levels of the enzymes in the plant.
Another embodiment of the invention is to express the E1 cellulase (endo-1,4-β-D-glucanase, EC 3.2.1.4) and CBH I (cellobiohydrolase or 1,4-β-D-glucan cellobiosidase, EC 3.2.1.91) in corn. In a further embodiment, the E1 cellulase is secreted to the cell wall, retained in the endoplasmic reticulum or targeted to the vacuole of a plant cell. Another embodiment provides for CBH I enzyme to be secreted to the cell wall or retained in the endoplasmic reticulum.
Other embodiments are to further improve expression of cellulases in plants by backcrossing transgenic plants containing the cellulase expressing gene into plants with good agronomic traits.
The objectives of this invention will become apparent in the description below. All references cited are incorporated herein by reference.